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A strain highly expressing alkaline pectinase and its construction and application

A high-efficiency expression and pectinase technology, which is applied in the field of genetic engineering, can solve the problems that the yield of alkaline pectinase cannot be further improved, and limit the industrial production of alkaline pectinase, and achieves the effect of high-efficiency expression.

Active Publication Date: 2019-08-06
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A comprehensive comparison of different hosts that can express alkaline pectinase shows that the protein expressed by Pichia pastoris is easy to purify and has high yield, but the overexpression of heterologous protein will lead to growth stress and cause the unfolded protein effect (UPR), resulting in alkaline pectinase The yield can not be further improved, which limits the industrialized production of alkaline pectinase

Method used

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  • A strain highly expressing alkaline pectinase and its construction and application
  • A strain highly expressing alkaline pectinase and its construction and application
  • A strain highly expressing alkaline pectinase and its construction and application

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Embodiment 1: Construction and identification of recombinant bacteria

[0031] Extract Pichia pastoris RNA, reverse transcribe it into cDNA, use cDNA as a template, design primers, obtain ERO1 and UBC1 genes by PCR, clone them into the expression vector pGAPZA, and obtain recombinant plasmids pGAPZA-ERO1 and pGAPZA-UBC1, Then, according to the isotail enzyme effect of Bgl Ⅱ and BamH Ⅰ, a double gene combination co-expression vector pGAPZA-ERO1-UBC1 was constructed (see attached figure 1 ), transform the recombinant vector pGAPZA-ERO1-UBC1 into Pichia pastorisGS115-pPIC9K-PGL (the alkaline pectinase gene whose nucleotide sequence is shown in SEQ ID NO.1 is connected to the expression vector pPIC9K, and then transformed into Pichia pastoris Red yeast host strain GS115), and the co-expression recombinant strain Pichia pastoris GS115PGL-ERO1-UBC1 was obtained through screening and identification.

[0032] Primers are as follows:

[0033] ERO1 upstream CAATTGAACAACTATTTCGA...

Embodiment 2

[0039] Example 2: Enzyme activity assay and protein electrophoresis of co-expressed genetically engineered strains

[0040] Cultivation method: After seed activation, the strain was inoculated into the basic fermentation medium YPD, cultured at 30°C and 220rpm for 14h, then transferred to the optimized growth medium BMGY and cultured at 30°C and 220rpm for 24h, and then the strain Transfer to the induction medium BMMY at 23°C, 220rpm and add 1.5% methanol every 24h to induce the expression of alkaline pectinase.

[0041] The enzyme activity measurement conditions are: the fermentation broth is centrifuged at 8000rpm for 10 minutes, extracellular PGL is contained in the fermentation supernatant, and a certain amount is taken for detection. PGL reaction system: glycine-NaOH buffer containing 0.2% polygalacturonic acid (substrate) (0.2mol L -1 , 0.44mmol·L -1 CaCl2, pH9.4) 2mL, 20μL of the sample to be tested, and the inactive enzyme solution as the blank control. The PGL reac...

Embodiment 3

[0043] Embodiment 3: 3L fermentation tank fermentation culture

[0044] Pick a single colony from the solid medium plate and inoculate it in YPD medium (50ml liquid volume in a 500ml Erlenmeyer flask) at 30°C, and cultivate it at 220rpm for 24h as a seed solution, and then inoculate a 800ml batch fermentation culture with 10% inoculum base (85% phosphoric acid 26.7ml / L, CaSO 4 0.93g / L,K 2 SO 4 18.2g / L, MgSO 4 ·7H 2 O 14.9g / L, KOH 4.13g / L, glycerol 40.0g / L, PTM 1 4.35ml / L) in the 3L fermenter (NBS company of the United States), the initial stirring speed is 500r / min, the air flow is 2vvm, 50% ammoniacal liquor and 30% phosphoric acid control pH5.5, and the culture temperature in the growth period is 30 ℃, when Add 50% (w / v containing 12ml / L PTM 1 ) glycerol, when the glycerol was depleted of dissolved oxygen again, starvation cultured for 1 hour, and then began to add induction medium (100% methanol containing 12ml / L PTM 1 ), while the temperature was lowered to 22° C...

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Abstract

The invention discloses a strain for efficiently expressing alkaline pectate and construction and application thereof and belongs to the technical field of gene engineering. ERO1 and UBC1 genes of Pichia pastoris are combined and cloned and connected to Pichia pastoris expression vector pPGAZA using gene recombination technology and converted to strain Pichiapastoris GS115-pPIC9K-PGL to obtain strain GS115 / PGL-ERO1-UBC1 more efficient in expressing alkaline pectate than an original strain. By shaker fermentation compared with the art not using the method, enzymatic activity of the strain GS115-pPIC9K-PGL is increased by 49.4%, with a significant increase; in fermentation culture in a 3L fermentation tank, the recombinant strain GS115 / PGL-ERO1-UBC1 is up to 1362.31 U / ml in maximum enzymatic activity, efficient expression of the alkaline pectate is achieved, and good basis is laid for the large-scale production of the alkaline pectate.

Description

technical field [0001] The invention relates to a strain highly expressing alkaline pectinase and its construction and application, belonging to the technical field of genetic engineering. Background technique [0002] Pectinase is a complex enzyme that breaks down pectin polymers into unsaturated oligogalacturonic acids. The enzyme is widely distributed and found in some parasitic nematodes, plants and microorganisms. Pectinase is widely used and has a history of industrial application for more than 40 years. Pectinases are divided into acid pectinases and alkaline pectinases PGL according to the optimum reaction pH. Among them, acid pectinase is mainly used in clarification of fruit juice and wine, extraction of fruit and vegetable juice, fruit peeling and so on. PGL applications are mainly used in textile, food, paper industry and environmental fields. The application of enzymatic method to the above-mentioned field-related reactions has the advantages of environmenta...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/19C12N9/88C12R1/84
CPCC12N9/88C12Y402/02002
Inventor 刘松陈双全陈坚堵国成
Owner JIANGNAN UNIV
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